This invention relates to densitometers particularly adapted for detection and quantification of molecules separated by gel electrophoresis using photothermal techniques.
Electrophoresis is an analytical technique widely used by medical researchers, biochemists and molecular biologists for characterization of large atomic weight charged molecules, such as proteins and nucleic acids.
In one type of electrophoresis procedure, a sample to be characterized is deposited at an area of a slab of gel material, made for example, from a polyacrylamide which provides a matrix through which the sample molecules can migrate. The gel is exposed to a DC electrical field which causes charged molecules of the sample to migrate through the gel. The gel material inherently traps smaller molecules less efficiently than larger molecules. Accordingly, after a period of exposure to the electrical field, molecules are deposited at certain regions of the gel in accordance with their moledular weight, size, structure and charge.
Most of the molecules of interest for characterization by electrophoresis procedures are transparent in the visible and near ultraviolet range. Their ultraviolet absorption is masked by the absorption of constituents of the gel medium. Therefore, once the molecules have migrated within the electrophoresis gel, they are stained to permit their detection by optical techniques. Presently, transmission-type densitometers are used to characterize the stained areas of the gel. In these devices, light from a low power laser, or tungsten or deuterium lamp is passed through the sample and the absorption of the light caused by the stained spot on the gel is evaluated. Absorption is determined simply by the difference between detected light with and without the sample interrupting the light beam. Direct transmission-type densitometers have inherent limitations in their detection limits which are a function of optical noise and detector sensitivity.
Due to the sensitivity limits of conventional transmission-type densitometers, the staining procedure must produce an optically dense patch on the gel sample. In one present staining procedure, the gel is soaked with silver nitrate and the ionic silver is reduced to metallic silver which is deposited in the areas where the organic molecules are present. For many classes of organic molecules, silver staining techniques result in optically dense patches appearing on the electrophoresis gel which can be quantified using conventional light transmission techniques. Although silver staining of electrophoresis gels provides meaningful data, the procedure suffers from several disadvantages. Silver staining procedures are tedius, time consuming and complex. The reagents employed for silver staining are furthermore relatively expensive. Moreover, silver stains cannot be used to stain all types of molecules.
Another technique for imaging patches of organic molecules on electrophoresis gels is autoradiography. This procedure produces high optical density on photographic film contacting the gel regions in the areas of various classes or organic molecules. Autoradiography procedures are even more complex and tedious than silver staining. The procedure is however, presently required for characterizing molecules which cannot be stained using other procedures to a level within the detection limits of conventional transmisson densitometers.
Still another staining technique for electrophoresis gels uses Coomassie Brilliant Blue dyes. These dyes are inexpensive and simple to apply. Unfortunately, the optical density produced by Coomassie Brilliant Blue dyes for some samples is not sufficient to generate adequate absorption for existing transmission types densitometer devices, and accordingly, the use of such dyes has been limited in the past where high sensitivity is required.
Accordingly, there is a need to provide a densitometer apparatus for reading electrophoresis gels which provides higher sensitivity, thus facilitating simplified gel staining procedures.